The oligosaccharide moiety of glycoconjugates play important roles in several biological processes of a cell, including the folding and transport of glycoproteins across cellular compartments. For the biosynthesis of these complex oligosaccharides an intricate machinery exists in a cell. Defective glycan synthesis has serious pathological consequences and results in several human diseases. The oligosaccharide moieties bind to cellular proteins with high specificity and modulate the homo- and hetro-dimerization of glycoproteins. Due to the conformational flexibility of oligosaccharides, the torsional angles of a disaccharide unit, especially around the alpha-1-6-linkage, adjust in such a way that the side groups of the oligosaccharides orient themselves in a manner that promotes favorable interactions with the binding residues of the protein. Branched oligosaccharides cross-link proteins and generate infinite networks of protein-carbohydrate complexes, resulting in the modulation of various cell responses. In humans b4Gal-T1 family members are responsible for the synthesis of Gal moiety in different oligosaccharides, indicating that although all these enzymes transfer Gal to GlcNAc, each recognizes the remaining oligosaccharide moieties to which GlcNAc is attached differently. The sequence comparison of the human b4Gal-T family members reveal only a little or no variation among the family members in the GlcNAc binding site where as the extended oligosaccharide binding region shows significant variations, indicating that these enzymes may prefer different GlcNAc containing oligosaccharides as their preferred sugar acceptors. To determine the exact mode of binding of the oligosaccharide in the binding site we have carried both MD simulations as well as crystal structure analysis of the b4Gal-T1-oligosaccharide complexes. Defining the oligosaccharide binding site of b4Gal-T1 by docking oligosaccharides into the binding site and by crystal structure investigation of the complexes with the oligosaccharides : We have continued to use molecular modeling methods to study the binding of oligosaccharides to proteins, in particular the binding of various oligosaccharide substrates to b4Gal-T1, the 3D-structure of which has been determined in our laboratory, either in complex with UDP-galactose and Mn2+ ion, or in complex with alpha-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 09305-08 LECB). Examination of the GlcNAc binding site in b4Gal-T1 from the crystal structure reveals an "open canal shaped" extended sugar binding site that lies behind the GlcNAc binding site. This site is formed by the residues from three regions; residues 280 to 289, residues 319 to 325 and residues 359 to 368. LA binds to this region in the crystal structure of b4Gal-T1-LA complex, therefore it is expected to compete with the GlcNAc containing oligosaccharides such as chitobiose. These modeling studies have shown, which have concurred that among the different GlcNAc containing disaccharides only beta-linked disaccharides such as GlcNAc-beta-1,4-GlcNAc or GlcNAc-beta-1,2-Man are preferred over alpha-linked disaccharides. In fact alpha-methyl-GlcNAc is less preferred compared to GlcNAc by itself. Crystallization of the wild type b4Gal-T1with the acceptor either in the presence or absence of UDP has not been successful so far. This is mainly due to the absence of the acceptor binding-site in the apo-b4Gal-T1 that exists in the open conformation. The enzyme has been crystallized in the closed conformation, where the acceptor site is present, only when UDP-Gal is bound. Although UDP or the acceptor molecules can induce the essential conformational changes, such complexes have been crystallized thus far only in the presence of LA. Since LA binds to the extended sugar binding site it is not possible to crystallize b4Gal-T1 with the oligosaccharide acceptors in the presence of LA.